DNA Ladder Research Articles

Evaluation of pro-apoptotic potential of taxifolin against liver cancer.

Liver cancer is the second most common cause of cancer-induced deaths worldwide. Liver cirrhosis and cancer are a consequence of the abnormal angio-architecture formation of liver and formation of new blood vessels. This angiogenesis is driven by overexpression of hypoxia-inducible factor 1-alpha (Hif1-α) and vascular endothelial growth factor (VEGF). Apart from this, protein kinase B (Akt) is also impaired in liver cancer. Despite the advancement in conventional treatments, liver cancer remains largely incurable. Nowadays, the use of naturally occurring anticancer agents particularly flavonoids is subject to more attention due to their enhanced physicochemical properties. Therefore, this study underlines the use of a natural anticancer agent taxifolin in the treatment of liver cancer using hepatocellular carcinoma cell line HepG2 and Huh7. The aim of our study is to devise a natural and efficient solution for the disease prevalent in Pakistan. The study involved the assessment of binding of ligand taxifolin using molecular docking. The binding of taxifolin with the proteins (Hif1-α, VEGF and Akt) was calculated by docking using Vina and Chimera. Further evaluation was performed by cell viability assay (MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) Assay), colony formation assay, cell migration assay, DNA ladder assay and flow cytometry. To see whether taxifolin directly affected expression levels, analysis of gene expression of Hif1-α, VEGF and Akt was performed using real-time polymerase chain reaction (qPCR) and western blotting. In silico docking experiments revealed that these proteins showed favorable docking scores with taxifolin. Treatment with taxifolin resulted in the inhibition of the liver cancer growth and migration, and induced apoptosis in HepG2 and Huh7 cell lines at an inhibitory concentration (IC50) value of 0.15 µM and 0.22 µM, respectively. The expression of HIF1-α, VEGF and Akt was significantly reduced in a dose- dependent manner. The inhibitory effect of taxifolin on hepatic cells suggested its chemopreventive and therapeutic potential. The studied compound taxifolin exhibited pronounced pro-apoptotic and hepatoprotective potential. Our study has confirmed the pro-apoptotic potential of taxifolin in liver cancer cell lines and will pave a way to the use of taxifolin as a chemotherapeutic agent after its further validation on the animal models and humans based epidemiological studies.

Comparison and improvement of DNA extraction methods in Buxus hyrcana

Buxus hyrcana is one of the endangered and evergreen species of the Hyrcanian forests in Iran. The genetic diversity assessment is an essential step towards the conservation of this species. High-quality DNA is required for molecular markers analysis; therefore we compared different DNA extraction methods on leaf samples of B. hyrcana. The quantity and quality of the extracted DNAs were evaluated by spectrophotometry and gel electrophoresis. Also, ISSR (Inter simple sequence repeats) markers were applied on the extracted DNAs to compare their quality for PCR amplification. Results showed that quantity, quality, and PCR efficiency and reproducibility were different for DNA extracted using different methods. The quality of the DNA at the absorbance A260/A280 ratio ranged from 1.02 to 1.97. The highest concentration of DNA measured by spectrophotometry belonged to the Cota-Sanchez extraction protocol (695.3 ng/l) and the lowest value was obtained with Edward4 method (204.7 ng/l). The modified Onate method (Onate2) was extracted the highest DNA concentration by comparison of brightness against the DNA ladder. Among the different extraction methods, the good quality and quantity were obtained in extracted DNA for Doyle and Doyle, Cota-Sanchez and modified Onate protocols; the latter method (Onate2) created both good quality and quantity of extracted DNA and operated effectively in terms of cost and time. Onate2 had the best amplification results with ISSR primers.

Corrigendum] Cyclophilin D modulates cell death transition from early apoptosis to programmed necrosis induced by honokiol

Subsequently to the publication of the above article, the authors have realized that the printed version of Fig.6 on p.1661 contained some mistakes. Potential anomalies regarding this figure concerning the duplication of data both within Fig.6 and comparing data between Figs.5 and 6 were also drawn to our attention by an interested reader. Specifically, the authors realized that the bands of BCL‑xl were erroneously selected from the GAPDH dataset during the process of compiling this figure. The authors subsequently found that the original photographs of these western blot bands had been lost during the time period that had elapsed since these experiments were completed. In order to corroborate the results, the authors repeated the contentious experiments shown in Fig.6 and obtained similar results, thereby corroborating the results and conclusions reported in this study. A revised version of Fig.6, containing the newly obtained data, is shown below. The errors made with the assembly of Fig.6 originally did not have an adverse bearing on the overall conclusions reported in this study. The authors are grateful to the Editor of International Journal of Oncology for allowing them the opportunity to publish this Corrigendum, and all of the authors agree to the publication of this Corrigendum. The authors sincerely apologize for their errors, and apologize to the readership of the Journal for any inconvenience caused. [the original article was published in International Journal of Oncology 42:1654‑1663, 2013; DOI: 10.3892/ijo.2013.1863].

Serial profiling of cell-free DNA and nucleosome histone modifications in cell cultures

Recent advances in basic research have unveiled several strategies for improving the sensitivity and specificity of cell-free DNA (cfDNA) based assays, which is a prerequisite for broadening its clinical use. Included among these strategies is leveraging knowledge of both the biogenesis and physico-chemical properties of cfDNA towards the identification of better disease-defining features and optimization of methods. While good progress has been made on this front, much of cfDNA biology remains uncharted. Here, we correlated serial measurements of cfDNA size, concentration and nucleosome histone modifications with various cellular parameters, including cell growth rate, viability, apoptosis, necrosis, and cell cycle phase in three different cell lines. Collectively, the picture emerged that temporal changes in cfDNA levels are rather irregular and not the result of constitutive release from live cells. Instead, changes in cfDNA levels correlated with intermittent cell death events, wherein apoptosis contributed more to cfDNA release in non-cancer cells and necrosis more in cancer cells. Interestingly, the presence of a ~ 3 kbp cfDNA population, which is often deemed to originate from accidental cell lysis or active release, was found to originate from necrosis. High-resolution analysis of this cfDNA population revealed an underlying DNA laddering pattern consisting of several oligo-nucleosomes, identical to those generated by apoptosis. This suggests that necrosis may contribute significantly to the pool of mono-nucleosomal cfDNA fragments that are generally interrogated for cancer mutational profiling. Furthermore, since active steps are often taken to exclude longer oligo-nucleosomes from clinical biospecimens and subsequent assays this raises the question of whether important pathological information is lost.

BPA toxicity during development of zebrafish embryo.

Bisphenol A (BPA) is a monomer used in the production of polycarbonate, a polymer commonly found in plastics, epoxy resins and thermal papers. The presence of BPA in food, water, air and dust has been of great concern in recent years not only due to environmental and ecological issues but also because of its supposed risk to public health related to its mutagenic and carcinogenic potential. In this study we evaluated the toxicity of bisphenol A in zebrafish embryos (Danio rerio) and determined the 50% lethal concentration (LC50) of this chemical. BPA was used at concentrations ranging from 1 μM to 100 μM in E3 medium/0.5% dimethylsulfoxide (DMSO) from previously prepared stock solutions in 100% DMSO. Controls included embryos exposed only to E3 medium or supplemented with 0.5% DMSO. Camptothecin (CPT), a known inhibitor of cell proliferation was used as positive control at a concentration of 0.001 μM in E3 medium/0.5% DMSO. Adults zebrafish were placed for breeding a day before the experimental set up, then, viable embryos were collected and selected for use. Experiments were carried out in triplicates, according to specifications from Organization for Economic Cooperation and Development (OECD). One embryo/well (25 embryos per concentration) was distributed in 96 well microplates in presence or absence of the chemicals. The plates were kept in BOD incubators with a controlled temperature of 28.5 ºC and with photoperiod of 14 h light:10 h dark. After 24h, 48h, 72h and 96h exposure, the exposed embryos were evaluated according to the following parameters: mortality, coagulation, rate of heartbeat, hatching and presence of morphological abnormalities. Photography was obtained by photomicroscopy. Apoptosis was evaluated by DNA ladder assay. DNA was extracted by phenol:chloroform method and analyzed by 2% agarose gel electrophoresis. DNA fragments were visualized after ethidium bromide staining in ultraviolet transilluminator. The LC50 determined for BPA was 70 μM after 24 hours, 72 μM after 48 hours, 47 μM after 72 hours and 31 μM after 96 hours exposure. BPA induced morphological and physiological alterations such as yolk sac and pericardial edema, hatching delay or inhibition, spine deformation, decreasing in heartbeat rate and mortality. In conclusion, this study demonstrated that BPA induced marked malformations in zebrafish embryos at concentrations above 25 μM corroborating the current concerns related to the widespread presence of BPA in the air, food and water used by humans as well as in the bodily fluids and tissues.

THE IMPACT OF ISCHEMIA-REPERFUSION ON LANGENDORFF RABBIT HEART WITH EXPERIMENTAL ATHEROSCLEROSIS

Abstract Objective: To examine the effect of hypercholesterolemia (HC)on cardiac function in rabbit model. Design and method: 12 adult rabbits divided into 2 groups of 6 rabbits each:Control(C) fed on normal chow, and (HC) fed on 2% cholesterol (Iard mixed with chow) for 16 weeks. After treatment, rabbits from (C) and (HC) groups were sacrificed and hearts were quicky removed, and perfused with standard Krebs Hanseleit buffer at a rate of 10 ml/minute at 37°C, pH 74 in Langendorff retrograde perfusion system at a constant pressure.45 minutes ischemia was followed by 120 minutes reperfusion and parameters of cardiac function: coronary flow (HC), Heart rate (H.R.) and left ventricle developed pressure (LVDP) of hearts from (C) and (HC) groups were measured. TACS DNA laddering kit was used to assess left ventricle cardiomyocytes from group (HC) for apoptosis, also fragments of left ventricle from (HC) were processed for Electron Microscopy (EM). Results: Evaluation of (C.F.), (H.R.) and (LVDP) in (C) and (HC) groups have pointed out a 50% reduction in (C.F). in (HC) group versus (C) during stabilization period In (HC) group, LVDP was depressed during the whole experiment, while in (C) group is was almost constant. Protection of myocardium against oxidative stress was significantly depressed following (HC) treatment, synthesis and utilization of GSH was limited versus (C). Cardiac hypertrophy experimentally induced with (HC) was related with a decrease in (C.F.) and LVDP associated with an increase in lipid peroxidase, in LDH and CK as markers of myocardial lesion and GGT activity in myocardial tissue. Myocyte apoptosis was present in (HC) group, pointed out by DNA laddering pattern on gel electrophoresis. (E.M.) studies have pointed out perinuclear edema of myocytes, dilated sarcoplasmic reticulum and swollen mitochondria. Conclusions: High cholesterol diet in (HC) rabbits induces cardiac hypertrophy and changes in physiological parameters of heart with a negative impact upon synthesis of antioxidant enzymes as well as changes in ultrastructure of heart muscle and at the molecular level (DNA laddering is present as a sign of apoptosis in ventricular myocytes).

A Simple PCR-Based Method for the Preparation of DNA Size Markers

DNA size markers are widely used in molecular biology laboratories as DNA molecular weight standards. In this paper, I report a simple and convenient method for the preparation of DNA ladders using PCR technique. DNA ladders usually range from 100 bp to 10 kb, and many such 100 bp and 1 kb DNA ladders are commercially available. Conventionally, 100 bp to 5 kb DNA fragments were amplified using standard PCR with Taq DNA polymerase, and the large DNA fragments exceeding 5 kb were amplified using long-range PCR with a special thermostable polymerase. In this study, several types of DNA ladders were prepared by mixing the amplified DNA fragments in different combinations, which can be used as DNA size standards in agarose gel electrophoresis.

Stachyose triggers apoptotic like cell death in drought sensitive but not resilient plants

Programmed cell death (PCD) is one of the most intensively researched fields in modern mammalian biology with roles in cancer, aging, diabetes and numerous neurodegenerative diseases. It is becoming increasingly clear that PCD also plays significant roles in plant defence and responses to the environment. Given their unique ability to tolerate desiccation (cells remain viable even after they’ve lost 95% of their water), resurrection plants make ideal models to study the regulation of plant PCD pathways. Previously, we showed that the Australian resurrection plant, Tripogon loliiformis, suppresses plant PCD, via trehalose-mediated activation of autophagy pathways, during drying. In the present study, we created a full-length T. loliiformis cDNA library, performed a large-scale Agrobacterium screen for improved salinity tolerance and identified Stachyose synthase (TlStach) as a potential candidate for improving stress tolerance. Tripogon loliiformis shoots accumulate stachyose synthase transcripts and stachyose during drying. Attempts to generate transgenic plants expressing TlStach failed and were consistent with previous reports in mammals that demonstrated stachyose-mediated induction of apoptosis. Using a combination of transcriptomics, metabolomics and cell death assays (TUNNEL and DNA laddering), we investigated whether stachyose induces apoptotic-like cell death in T. loliiformis. We show that stachyose triggers the formation of the hallmarks of plant apoptotic-like cell death in the desiccation sensitive Nicotiana benthamiana but not the resilient T. loliiformis. These findings suggest that T. loliiformis suppresses stachyose-mediated apoptotic-like cell death and provides insights on the role of sugar metabolism and plant PCD pathways. A better understanding of how resilient plants regulate sugar metabolism and PCD pathways may facilitate future targeting of plant metabolic pathways for increased stress tolerance.

The Effect of hsa-miR-451b Knockdown on Biological Functions of Gastric Cancer Stem-Like Cells.

Numerous researches have extensively studied factors such as microRNAs that lead to cancer. Thus, the current study's purpose is to investigate the biological consequences of hsa-miR-451b inhibition on the properties and functions of gastric cancer stem-like cells. First, gastric cancer stem-like cells were transfected by hsa-miR-451b inhibitor then we used real-time RT-PCR to evaluate its effect on the expression of hsa-miR-451b and two of its direct target genes, Stemness markers such as KLF4, SOX2, CD44, OCT3/4 and NANOG genes and finally Akt, PI3K, Bcl-2, Bax, CASP3 and PCNA genes involved in apoptosis. Here, we conducted a DNA Laddering assay to investigate apoptosis.The level of the MMP-2 and -9 Activities and Migration were examined by Zymography and Transwell invasion assay. HUVEC cells were used to investigate angiogenesis. The outcomes revealed that the level of the MMP-2 and -9 Activities, migration and angiogenesis decreased, but apoptosis was induced by inhibiting hsa-miR-451b. Evaluating KREMEN1 and CASK expression showed that the former increased, and the latter dropped under hsa-miR-451b inhibition. Also, upregulation of the KLF4 and SOX2 and downregulation of the CD44, OCT3/4, and NANOG decreased Self-renewal ability of gastric cancer stem cells under hsa-miR-451b inhibition. Even, under hsa-miR-451b inhibition, downregulation of Akt, PI3K, Bcl-2 and PCNA as well as upregulation of Bax and CASP3 revealed a movement towards apoptosis in MKN-45 stem-like cells. In summary, hsa-miR-451b is an oncomir in the carcinogenesis of gastric cancer stem-like cells and may be suggested as an appropriate therapeutic target for future gastric cancer treatment.

The combination of berberine and methotrexate enhances anti-cancer effects in HeLa cancer cell line: A morphological study

Background: Co-administration of two or several either chemotherapeutic agents or conventional drugs as a combination treatment is the most effective method to increase therapeutic efficiency. Additive or synergistic influence are two mechanisms by which combination therapy causes a rise in optimal cancer therapy compared to a single treatment. Methods: Colorimetric assay was carried out to estimate the cytotoxicity of the combined system, followed by apoptosis assay to calculate the number of apoptotic cells. Both 4′,6-diamidino-2-phenylindole (DAPI) staining and DNA ladder were complemented tests to illuminate morphological changes and DNA fracturing on HeLa cancer cells. Statistical: Through Graph pad Prism 6.0 software. One-way ANOVA was used to determine the significance. A P-value of less than 0.05 was considered to be statistically significant. Results: In this study revealed that the combination of MTX and BER could inhibit the growth of HeLa cancer cells noticeably. Nevertheless, single BER and MTX were not as effective as a combined system to reduce cell viability at the same dose. Regarding the apoptosis induction and change in morphology of cancer cells’ nucleus, co-treatment of BER and MTX was more effective. The result was complemented with flow cytometry, DAPI staining and DNA ladder, which showed that BER+MTX depicted more anti-cancer effects. Conclusion: The combination therapy of HeLa cancer cells with BER and MTX showed high inhibition effect compared to other treated groups.

Convalescent blood plasma (CBP) donated by recovered COVID-19 patients – A comment

Convalescent blood plasma (CBP) donated by recovered COVID-19 patients – A comment

The Effect of Indian Fig Fruit Extract on Human Papilloma Virus containing Cervical Cancer Cells (HeLa) by Decreasing the HPV18 L1 Gene Load.

Background:Global trend is moving towards the use of natural phytochemicals to fight against pathogens. Human cervical cancer is directly associated with onco-potent type of Human Papilloma Virus (HPV). There is no known medicine for clearance of HPV type whose persistence is the cause of occurrence and re-occurrence of cervical cancer. The different species of fig fruit and their latex are reported to have HPV associated genital warts clearance capability.Methods:In the current investigation, the effect of the methanol extract of Ficus benghalensis L. fruits on HPV type18 viral load in HeLa cell line was tested by doing PCR using HPV L1 primers (MY09/My011) and the cytotoxicity was also analysed by MTT assay. The induction of apoptotic activity in terms of DNA fragmentation and hyper-chromic effects of DNA was analysed. Results:The PCR results showed a reduction in the HPV18 DNA and also the treatment exhibited a promising cytotoxicity with IC50 value at 211.86 μg/ml. The DNA samples from treated HeLa cells showed DNA shearing and laddering as a mark of apoptotic DNA fragmentation (Fig. 2) and the UV absorbance value at 260 nm was found to be significantly (P<0.01) higher in the DNA sample treated with fruit extract compared to the untreated DNA sample. Conclusion:The Ficus benghalensis L. fruit extract reduced the HPV viral load in HPV18 containing HeLa cells and showed an effective cytotoxicity on HeLa cell line. It also could induce the apoptotic activity in HeLa cell line and this study results suggest that the Ficus benghalensis L. fruits can be used to fight against cervical carcinoma, acting on HPV load.

A Highly Cellular Uptake Ternary Nanocomposite Titanate Nano-Tubes/CuFe₂O₄/Zn-Fe Could Induce Intrinsic Apoptosis of Prostate Cancer Cells: An Extended Study.

Our previously prepared ternary nanocomposite TNT/CuFe₂O₄/Zn-Fe was highly engulfed by PC-3 cells, activated cytotoxicity that was dosage and time-subordinated, and demonstrated morphological alteration, which is one of the common characteristics of apoptotic cells. This prolonged study aimed to investigate other items. The study performed assays as Annexin V-FITC, flow cytometry, DNA ladder electrophoresis, and ROS assay for apoptosis detection, cell cycle analysis, DNA fragmentation, and ROS generation, respectively. In the PC-3-treated cells, the early and late phases of apoptosis with different percentages and DNA fragmentation were determined. Besides, the PC-3 cell cycle revealed the three major cell distribution different phases of the cycle (G1, S, and G2/M), and the Sub G1, which corresponded to apoptotic cells. The results proved the presence of ROS that triggered the intrinsic apoptotic pathway, which was confirmed through a decrease in (Bcl-2), the release of cytochrome c, activation of caspase-9, and caspase-3. To conclude, the ternary nanocomposite TNT/CuFe₂O₄/Zn-Fe achieved biochemical features alterations and could induce intrinsic apoptosis of PC-3 cells. The planned work of the current research will illuminate the arrested phase in the cell cycle through studying tumor suppressor genes such as p53 and Retinoblastoma RB, c-Myc oncogene, and cyclin-dependent kinases (Cdks) as well as their regulators.

Ameliorative effects of high-protein diet on hepatotoxic alterations in Swiss albino mice exposed to mobile phone radiation

Objectives: Nowadays, mobile phones have become a prime necessity. At present, various advance networking connections such as 3G and 4G are available in the market to get a faster service. However, these mobile phones emit harmful electromagnetic radiations which have various ill effects on different physiological systems. The present study has been done to find out the extent of damage caused by mobile phone radiation on liver cells, and, whether high-protein diet (HPD) has any ameliorative effect on such changes. Materials and Methods: Male Swiss Albino mice were divided into four groups. Two groups of animals were exposed to mobile phone radiation at global system for mobile communications like frequencies at 1.8 GHz for 3 h continuously per day for 90 days. One of these radiation exposed groups received a normal diet (containing 5% casein) and the other exposed group received a HPD (supplemented with 20% casein). The control group animals received only a normal diet, while the animals of the remaining group received only a HPD. The serum biochemical and histological parameters of liver tissues and ladder assay of hepatic DNA were studied. Results: A significantly elevated level (P &lt; 0.05) of aspartate aminotransferase, alanine transaminase, bilirubin and serum caspase-3 levels was observed in the group of animals exposed to electromagnetic radiation emitted from mobile phone. Histological changes were also observed in the experimental groups. Moreover, in genomic DNA ladder assay, fragmented DNA was observed in exposed group, which is a probable sign of cell apoptosis. Recovery symptoms were observed in the animals supplemented with high casein diet. Conclusion: From the present study, it may be concluded that electromagnetic radiation emitted from mobile phones has damaging effects on hepatocytes which may be ameliorated by HPD.

Retraction Note to: Effects of microRNA-21 on Nerve Cell Regeneration and Neural Function Recovery in Diabetes Mellitus Combined with Cerebral Infarction Rats by Targeting PDCD4.

We aimed to determine the effect and mechanism of microRNA-21 (miR-21) on nerve cell regeneration and nerve functional recovery in diabetes mellitus combined with cerebral infarction (DM + CI) rats by targeting PDCD4. A total of 125 male Wistar rats were selected for DM + CI rat model construction and assigned into the blank, miR-21 mimics, mimics control, miR-21 inhibitor, inhibitor control, miR-21 inhibitor + si-PDCD4 and si-PDCD4 groups. And, 20 healthy rats were selected for the normal group. Triphenylterazolium chloride (TTC) staining and HE staining were used for determination of the area of CI and pathological changes, respectively. Behaviors of rats in the eight groups were determined by forelimb placement test and balance beam walking test. Immunohistochemical staining, double immunofluorescence staining assay, Western blotting, and qRT-PCR were used to detect expressions of miR-21, PDCD4, HNA, Nestin, NeuN, β-III-Tub, PTEN, FasL, and GFAP. DNA laddering and TUNEL staining was used for cell apoptosis. TTC and HE staining confirmed that 87.5% rats were induced into CI + DM models successfully. Results of forelimb placement test and balance beam walking test showed that miR-21 mimics, and si-PCDC4 improved the nerve defect of model rats. Comparing with the blank group at the same time, rats in the miR-21 inhibitor group displayed significant decrease in the forelimb placement test score, significant increase in the balance beam walking test score, and exacerbation of nerve defect, while rats in the miR-21 mimics and si-PCDC4 groups displayed significant increase in forelimb placement test score and significant decrease in the balance beam walking test score and improvement of nerve defect situation. The HNA, Nestin, and PDCD4 expressions were decreased and the NeuN, β-III-Tub, and GFAP expressions were increased in the miR-21 mimics and si-PDCD4 groups comparing with the blank group. The results of miR-21 inhibitor group were on the contrary. In comparison to the blank group, the miR-21 mimics group and the si-PDCD4 had lower miR-21 expressions and higher expressions of PDCD4, PTEN, and FasL, while the miR-21 inhibitor group was in the opposite trend. The results of qRT-PCR were the same with Western blotting. The expressions of fluorescence in other groups were higher than the normal group; compared with the blank group, the miR-21 mimics group and the si-PDCD4 group had lower fluorescence expression and DNA ladder. However, the fluorescence expressions and DNA ladder of miR-21 inhibitor group increased markedly in contrast with the blank group. Comparing with the blank group, BrdU+/DEX+ fluorescence intensity significantly enhanced in the miR-21 mimics and si-PDCD4 groups and significantly reduced in the miR-21 inhibitor group. And, comparing with the blank group, in the miR-21 mimics group, the signal strength of luciferase carrying the wild-type PDCD4 was reduced by 25%. The present studies demonstrated that miR-21 could promote the nerve cell regeneration, suppress apoptosis of nerve cells in DM + CI rats and improves the nerve defect situation of DM + CI rats by inhibiting PDCD4.

Synthesis, characterization, in vitro biological and molecular docking evaluation of N,N'-(ethane-1,2-diyl)bis(benzamides)

The present research describes the synthesis, characterization, in vitro biological and docking evaluation of N,N'-(ethane-1,2-diyl)bis(benzamides) (3a-3j). Consequently, in in vitro hRBCs hemolysis assay, only the bis-amide (3d) induced 52.4% hemolysis at higher concentration (1000 μg/mL) that decreased drastically with concentration (250 μg/mL) to 27.9% (CC50 = 400.41). Similarly, the tested bis-amide (3j) was found to be the least toxic with 7.8% hemolysis at higher concentration (1000 μg/mL) that gradually decreases to 6.1% (CC50 = 19,347.83) at lower concentration (250 μg/mL). Accordingly, the tested bis-amides were found to be highly biocompatible against hRBCs at higher concentrations with much higher CC50 values (> 1000 μg/mL). The biocompatible bis-amides (3a-3j) were subjected to in vitro DNA ladder assay to analyze their apoptotic potential. The results obtained suggest the tested bis-amides (3a-3j) are highly degradative toward DNA causing the appearance of more than one bands or complete degradation of DNA except (3a), (3c), (3i) and (3 g). Moreover, the synthesized bis-amides (3a-3j) were tested in in vitro antileishmanial assay to unveil their leishmaniacidal potential. The results obtained clearly indicated that some of the tested bis-amides displayed good dose dependent response. The tested bis-amides were highly active at higher concentration (1000 μg/mL) against the leishmanial promastigotes and their % inhibitory potential decreased drastically with concentration (250 μg/mL). Consequently, at higher concentration (1000 μg/mL), the bis-amide (3f) caused 85% inhibition and was ranked as the most effective leishmaniacidal bis-amides followed by the bis-amide (3 g) with 73.54% inhibition of leishmanial promastigotes. However, in terms of their IC50 values, the best leishmaniacidal potential was displayed by the bis-amide (3f) followed by (3b), (3j) and (3 g) with IC50 values increasing in the order of 633.16, 680.22, 680.22 and 712.93 μg/mL, respectively. Molecular docking studies revealed that bis-amides having electron-donating groups showed good binding potential against antileishmanial target.

Assessment of Immunotoxicity and Oxidative Stress Induced by Zinc Selenium/Zinc Sulphide Quantum Dots

Although ZnSe/ZnS quantum dots (QDs) have emerged as apparently less hazardous substitute to cadmium-based QDs, their toxicity has not been fully understood. Huge levels of ROS production and associated difficulties comprise the underlying reason for nanomaterial toxicity in cells. This will cause both immunotoxicity and genotoxicity. In the current work, Zinc Selenium/Zinc Sulphide (ZnSe/ZnS) QDs was synthesized, characterized and analyzed for its role in oxidative stress induction in two cell lines (HepG2 and HEK) and Swiss Albino mice. ROS production and influence of catalase activity in ROS production measured by DCFHDA assay in both HepG2 and HEK cells after exposure to ZnSe/ZnS QDs. Assessment of nitrile radical formation carried out by griess reagent. Level of GSH is assessed as a marker for oxidative stress induced by QDs. Cell death induced after exposure to ZnSe/ZnS QDs investigated by Calcein AM-PI live dead assay. Apoptotic DNA ladder assay carried out for studying the potential of ZnSe/ZnS QDs to induce DNA fragmentation. In vivo bio-nano interaction was studied by exposing Swiss Albino mice to ZnSe/ZnS QDs via i.v. and i.p. injection. Antioxidant assays were carried out in brain and liver homogenates to study the oxidative stress. LPO, GSH, GPx, GR and SOD are considered as biomarkers for the stress analysis. Blood brain barrier (BBB) integrity also studied. Spleenocytes proliferation assay was carried out to study the immunotoxicity response. ZnSe/ZnS QDs do not induce visible oxidative stress upto a concentration of 50 μg/ml. Cell death occurs at higher concentration (100 μg/ml) caused by ROS production. Overall study apparently provide attentive information that ZnSe/ZnS QDs is not capable of eliciting any serious damages to liver and brain tissues which in turn substantiates its applicability in biomedical applications.

Disturbance of cellular homeostasis as a molecular risk evaluation of human endothelial cells exposed to nanoparticles

Even though application of nanoparticles in medicine seems to provide unique solutions for drug delivery and diagnosis diseases, understanding interactions between nanoscale materials and biological systems is imperative. Therefore, this study determined the effect of different types of nanoparticles (NPs) on human endothelial cells and examined the types of toxicity responses they can induce. Four different types of NPs were tested (PLA/MMT/TRASTUZUMAB, PLA/EDTMP, PLGA/MDP, and Pluronic F127 MICELLES), representing three putative areas of application: anticancer therapy, scintigraphy, and cosmetology. The experiments were performed on immortalized human umbilical vein endothelial cells (HUVEC-STs). Light contrast phase microscopy as well as cell viability assays showed that only Pluronic F127 MICELLES decreased the number of HUVEC-STs in contrast to PLA/MMT/TRASTUZUMAB, PLA/EDTMP, and PLGA/MDP NPs, which altered cell morphology, but not their confluency. The tested NPs induced not only DNA strand-breaks and alkali-labile sites, but also internucleosomal DNA fragmentation, visualized as a DNA ladder pattern typical of apoptosis. Moreover, generation of free radicals and subsequent mitochondrial membrane potential collapse showed the significance of free radical production during interactions between NPs and endothelial cells. High concentrations of NPs had different degrees of toxicity in human endothelial cells and affected cell proliferation, redox homeostasis, and triggered mitochondrial dysfunction.

Rotenone-induced necrosis in insect cells via the cytoplasmic membrane damage and mitochondrial dysfunction

Rotenone, a selective inhibitor of mitochondrial complex I, has been extensively studied on kinds of neuron and neuroblast in Parkinson's disease. However, little is known about the potential mechanism of this promising botanical insecticide upon insect cells. In the article, cell proliferation of two Lepidoptera cell lines, Spodoptera litura SL-1 cells and Spodoptera frugiperda Sf9 cells, were all inhibited by rotenone in a time- and dose-dependent manner. Typical necrotic characteristics of cell morphology and ultrastructure, such as plasma membrane collapses and organelle lyses, were all observed by transmission electron microscope and scanning electron microscope. Moreover, irregular DNA degradation was also detected by DNA gel electrophoresis and Hoechst 33258 staining, while the typical apoptotic feature, DNA ladder, hadn't been observed. Flow cytometric analysis showed that rotenone-induced cell death of Sf9 and SL-1 cells accompanied with the plasma membrane potential depolarization and mitochondrial membrane potential reduction. Furthermore, the activity of Na+-K+-ATPase was detected in our study. In conclusion, rotenone could cause necrosis but not apoptosis in insect cells through a mitochondrial- and plasmic membrane-dependent pattern, which shed a light on the rotenone-induced cytotoxicity on insects

Evaluation of chemo-preventive efficacy of Ficus religiosa latex extract by flow cytometry analysis and gene expression studies performed by RT-PCR in various cell lines

BackgroundAn extract of Ficus religiosa latex has been previously found to possess potent pharmacological activity with high antioxidant content phytochemical. The present research was conducted to investigate the chemo-preventive efficacy of latex extract on human breast adenocarcinoma MDA MB 231, human neroblastoma IMR 32, and human colorectal HCT 116 cell lines.ResultsThe results showed that the latex crude extract induced cytotoxicity in all the selected cell lines with IC50 value 4.8 ± 1.13 μg/ml against the IMR 32 cell line. The cell cycle analysis results indicated the arrest and accumulation of cells at G1 phase in case of MDA MB 231 cells and HCT 116 cells whereas in the case of IMR 32 cells the arrest was in G2/M phase. The clear bands of fragments observed in DNA ladder experiments showed that apoptosis is induced by extracts in the cell lines. This could be correlated with the gene level expression studies on selected pro-apoptotic (p53 and caspase-3) and anti-apoptotic (Bcl-2, AKT) genes, which got upregulated and downregulated, respectively.ConclusionBased on the experimental evidence, Ficus religiosa contains phytochemicals with potent antitumor activities.